Craniofacial cartilage from human stem cells through neural crest stem cells Birth defects or injuries to the face can compromise an individual's self image and interactions with other people. At present, it is difficult to reconstruct cartilaginous features (nose, ears) with plastic surgery techniques, and transplanted tissue is often rejected without immunosuppressants. An attractive potential treatment is to use induced-pluripotent (iPS) cells from patients to grow functional craniofacial cartilage (cartilage of the head and face) for transplantation. Many hurdles must be overcome before cartilage can be grown in culture and subsequently transplanted into patients, however. Current techniques that derive cartilage from stem cells do not produce craniofacial cartilage. Existing techniques derive structural cartilage directly from pluripotent stem cells or embryoid bodies (masses of partially differentiated cells). These systems suffer from incomplete differentiation, and from subsequent differentiation into bone or blood vessels, overshooting the targeted development of cartilage. This is because they are forming mesoderm-derived cartilage, which normally subsequently differentiates into bone to form the skeleton. We have discovered a novel method to specifically generate craniofacial cartilage from human embryonic stem cells (hESCs) through a neural crest stem cell (NCSC) intermediate. Since neural crest- derived cartilage is the normal developmental pathway specific to craniofacial cartilage, it is less likely to suffer from incomplete differentiation, more likely to be a terminal differentiation step, and therefore less likely to give rise to undesired tissues, or tumors. We propose to determine the cell signaling mechanisms that produce human chondrocytes (cells that make up cartilage) from NCSCs, to fully characterize the culture conditions for accelerated differentiation into craniofacial cartilage from NCSCs derived from hESCs and iPS cells, and to grow cartilage structures in three dimensional matrices. We will deliver a protocol that will lead to subsequent studies assess the functional viability of cultured cartilage upon transplantation. These studies are the necessary first steps towards the long term goal of three-dimensional printing of complex biological scaffolds for chondrocytes to build a nose, ear, or other cartilaginous structures of the head and face.